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Non-O157 Shiga toxin (stx)–producing Escherichia coli (STEC) is recognized as an important human diarrheal pathogen. Cattle are the principal reservoirs of STEC, although other animals can be carri...

Environmental survival time is important when evaluating adverse health outcomes from foodborne pathogens. Although outbreaks associated with manure-impacted irrigation or runoff water are relative...

Background:This research work focused on ascertaining the presence of virulence and antibiotic-resistant genes inEscherichia coli(E. coli) O157 and non-O157 recovered from drinking water sources.Methods:Identification ofE. coliO157 and non-O157 was carried out using standard serological and PCR techniques. Virulence genes (rfbO157,fliCH7,stx1,stx2,eaeandhlygenes) and antibiotic-resistant gene (BlaTEM) were detected using PCR method on selected isolates (n= 15) from different water sources which demonstrated multiple antibiotic-resistance in a previous study.Results:The serological identification result revealed that a total of 68 out of 382E. coliisolates, recovered in a previous work, were identified as a presumptiveE. coliO157. These included 19.1 %, 21.7 %, 33.3 %, 14.3 % and 9.1 % ofE. coliisolates from wells, boreholes, sachets, streams and pipe-borne respectively. Statistical analysis revealed that there was no significant difference in the frequency ofE. coliO157 from the different water sources (p> 0.05). Also, there was a statistically significant positive correlation between theE. coliisolates andE. coliO157 (Pearson’sr= 0.996). Detection of virulence and antibiotic-resistant genes showed that only 46.7 %, 33.3 %, 33.3 %, 93.3 %, and 66.7 % carriedrfbO157,fliCH7,stx1,stx2 andrpoSgene respectively. In contrast, all the isolates possessedhlyand BlaTEMgenes but none hadeaegene.Conclusion:The presence of one or combination of these genes in these isolates depicts their virulence and resistance nature.

The current investigation was carried out for 100 random sample of meat products represented by frozen beef burger, frozen kofta, minced meat and oriental sausage (25 each) gathered from various shops and retail stores in Menofya governorate. The gathered samples were subjected to bacteriological examination for the isolation and identification of non O157 E.coli and E.coli O157 by using Conventional and recent techniques as multiplex PCR. By the conventional method the incidence of E.coli in the tested samples of beef burger, kofta, minced meat and oriental sausage was 6(24%), 9(36%) , 5(20%) and 11(44%) respectively. Multiplex-PCR technique was applied on 10 random meat product samples (3 negative and 7 positive for the isolation of E.coli by conventional method). M-PCR technique was applied in order to detect stx1 and stx2. In this study ,the M-PCR give negative result with all tested food samples. This study clarified that multiplex PCR may give negative results due to inhibitors that can be found in microbial DNA solutions extracted from meat sample or due to inhibitors which added during processing of meat products .

This study aimed to detect presence of RpoS gene in Escherichia coli O157:H7 and non-O157 and investigate their survival pattern in different water treatment methods. A total of fifteen serologically and molecularly identified E. coli was selected from a previous work, out of which eight were Escherichia coli O157 and seven were E. coli non-O157. From among these, S30 and S89 identified isolates served as presentative E. coli O157:H7 and non-O157 respectively for survival studies. The water treatment methods used employed included: use of silver, lime, storage, acidification (low pH), high temperature and Moringa oleifera. Survival pattern of the test organisms under the influence of these methods were carried out using standard techniques. Molecular detection of stress response gene, RpoS, in the fifteen (15) test organisms was performed following manufacturer’s instruction. Results showed that for both test organisms, silver was bactericidal at high concentration while storage allows their survival up till 21 days though with a reduction in cfu. Both organisms showed low survival at pH 9 while E. coli O157:H7 and non-O157 could survive at pH 4 and 6 respectively. E. coli O157:H7 survived better than non-O157 at high concentration of lime. While both survived at low temperature, E. coli O157:H7 survive better at 60oC. Sunlight and chlorine showed mild and complete bactericidal action respectively with increased exposure time for both test organisms. Moringa oleifera was only effective at a reduced concentration on the two organisms. Detection of RpoS genes showed that only 66.7% carried the gene in them while 33.3% did not. Findings from this study show that the possession of stressed genes in bacteria causing waterborne disease could allow these organisms to survive water treatment methods adopted in many under developed countries or rural communities. This suggests a threat to health of these communities.

The objective of this study was to quantify the frequency, distribution, and variability of fecal shedding and super-shedding of Shiga toxin–producing Escherichia coli (STEC) serogroups O26, O45, O...

Aims: A great concern directed to non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes due to their public health importance. Detecting the existence, antimicrobial profiles, and virulence repertoire of different STEC serotypes from animals essential for human food are important. Study Design: This study aimed to investigate the presence of STEC in different hosts, the distribution pattern of stx1, stx2, eaeA, and hlyA genes encoding Shiga toxins 1 and 2, intimin, and enterohemolysin, respectively, and the antimicrobial resistance of the detected serotypes. Results: A total of 75 samples were collected, 20 fecal samples from broilers, 15 fecal samples from ducks, 20 beef samples, and 20 human urine samples. Escherichia coli was detected at a rate of 60/75 (80%) distributed as;17 (85%), 8 (53.3%), 15(75%), and 20 (100%) from broilers, ducks, human urine, and beef samples, respectively. There was a significant difference between the isolation rates of E. coli from different sources with p

This study examined the biofilm-forming ability of six non-O157 Shiga-toxin-producing Escherichia coli (STEC) strains: O116:H21, wzx-Onovel5:H19, O129:H21, O129:H23, O26:H11, and O154:H10 on stainless steel coupons after 24, 48, and 72 h of incubation at 22 °C and after 168 h at 10 °C. The results of crystal violet staining revealed that strains O129:H23 and O154:H10 were able to form biofilms on both the submerged surface and the air–liquid interface of coupons, whereas strains O116:H21, wzx-Onovel5:H19, O129:H21, and O26:H11 formed biofilm only at the air–liquid interface. Viable cell counts and scanning electron microscopy showed that biofilm formation increased (p < 0.05) over time. The biofilm-forming ability of non-O157 STEC was strongest (p < 0.05) at 22 °C after 48 h of incubation. The strongest biofilm former regardless of temperature was O129:H23. Generally, at 10 °C, weak to no biofilm was observed for isolates O154:H10, O116:H21, wzx-Onovel5:H19, O26:H11, and O129:H21 after 168 h. This study found that temperature affected the biofilm-forming ability of non-O157 STEC strains. Overall, our data indicate a high potential for biofilm formation by the isolates at 22 °C, suggesting that non-O157 STEC strains could colonize stainless steel within food-processing facilities. This could serve as a potential source of adulteration and promote the dissemination of these potential pathogens in food.

Shiga toxin producing Escherichia coli is a contaminant of food and water that causes a diarrheal syndrome followed by more severe disease of the kidneys and symptoms of the central nervous system in humans. The isolation of Shiga toxin producing Escherichia coli (STEC) from diarrheic and apparently health calves is difficult due to lack of differential phenotypic characteristics from nonpathogenic Escherichia coli. The improvement of molecular technology allows identification of both toxin and serogroup specific genetic determinants. In this study, 300 fecal samples from diarrheic and apparently healthy calves were screened for STEC using PCR targeting Shiga toxin determinants. In addition routine culture methods for isolating O157 and non O157 STEC were also performed. The screening assays of serotyping isolates revealed 7 (4.1%) of O157H7, 156 (92.8%) of non O157 and 5 (3.1%) for untypable strains. These included STEC serotypes of O157H7 and O26 from diarrheic samples, and O78, O55 and O126 from apparently healthy calves. The high rate of STEC isolation and the diversity of STEC serogroup from calves focus the light on the importance of calves as reservoir of E. coli as well as motivate us to improve biosecurity measures in dairy farms.

From January to December 2016, Alberta Agriculture and Forestry (AAF) conducted a provincial survey of selected pathogens and indicator organisms on beef carcasses processed at Provincially Licensed Abattoirs (PLAs) in Alberta. The survey was conducted in seven small and medium scale slaughterhouses located in southern and northern Alberta that process beef cattle and cows. Paired samples were collected from the same carcass immediately after hide removal (pre-evisceration n = 401) and at pre-chill (n = 402) after application of a carcass wash and/or anti-microbial interventions. Swab samples were screened for the presence of Shiga toxin-producing Escherichia coli (STEC) O157:H7, six non-O157 STECs (O26, O45, O103, O111, O121 and O145), and Salmonella. In addition, samples were enumerated for indicator organisms (aerobic colony count (ACC), Enterobacteriaceae, coliforms, and generic E. coli). At pre-evisceration, 30 samples (7.4%) were confirmed positive for E. coli O157:H7; 13 samples (3.2%) were confirmed positive for non-O157 STEC; and 7 samples (1.7%) were confirmed positive for Salmonella. Pre-chill swabs had 21 samples (5.2%) positive for E. coli O157:H7, 16 samples (3.9%) positive for non-O157 STEC, and 1 sample (0.2%) positive for Salmonella. At pre-chill the prevalence of Salmonella was significantly lower (P

Lactoferrin has amajor effects on enteropathogenes as it inhibits growth. so, This work adopted to study the antibacterial activity of lactoferrin on growth of E. coli non O157 in broth and pasteurized milk. two strains of E. coli ( O1 carry stx2 and hly gene and O26 carry hly gene) were used. Different concentrations of lactoferrin (zero, 0.5, 1.0, 5.0, 10 and 20 mg/ml) were used in Lauria broth. Based on our results, LF showed various inhibition activity on E. coli non O157 growth in Lauria broth. Significant decrease observed on growth of E. coli O1. Higher concentrations of lactoferrin caused a significant decrease in E. coli O26 growth. Regarding its effect in pasteurized milk, a significant decrease in the count of E. coli O1and E. coli O26 at the concentrations of 10 and 20 mg/ml lactoferrin was noticed. So, lactoferrin could become a promising method to decrease growth of E. coli non O157 in pasteurized milk consequently decrease E.coli non O157 associated illness in humans.

The objectives of the current study were detection and characterization of the isolated Escherichia coli non O157 using PCR assay. A total of 90 cheese samples high salt soft cheese, Kareish cheese and Tallaga cheese were examined for E. coli non O157 using modified vancomycin- trypticase soy broth and Sorbitol MacConkey agar plates. Serodiagnosis of E. coli non-O157 has been done using slide agglutination test. Toxigenic E. coli non-O157 isolates were detected using PCR assay. Results postulated that the detection rate of non O157 using biochemical technique was 63.33% in Kariesh cheese, 20% in high salt soft cheese cheese, and 33.33% in Tallaga cheese. Ten different serotypes of E. coli non O157 have been distributed as following O1 (4.44%), O18 (1.11%), O20 (1.11%), O25 (1.11%), O26 (3.33%), O125 (1.11%), O126 (1.11%), O127 (1.11%) and untyped E. coli (18.89%). Regarding PCR results, serotypes group (O1) was positive for Stx2 gene, (O1 and O20) were positive for both Stx2 and hly genes, one serotype group O25 was positive for eaeA gene, one serotype group O26 was positive for hly gene while no detection for Stx1and STa genes. From the aforementioned data, attention must be paid to the problems of E. coli non O157 in foods. Consequently, more restriction and preventive measures should be taken in milk herds, milk production and dairy factories in respect to quality control sanitation and health care.

Background The implementation by diagnostic laboratories in England of polymerase chain reaction (PCR) to screen faecal specimens for Shiga toxin-producing Escherichia coli (STEC) has resulted in a significant increase in notifications mainly due to non-O157 strains. The purpose of this study was to develop an approach to public health risk assessment that prioritizes follow-up to cases caused by haemolytic uraemic syndrome (HUS) associated E. coli (HUSEC) strains and minimizes unnecessary actions. Methods Epidemiological and microbiological data were prospectively collected from 1 November 2013 to 31 March 2017 and used to compare three risk assessment approaches. Results A history of HUS/bloody diarrhoea/age under 6 years and faecal specimens positive for stx-predicted HUSEC with a diagnostic accuracy of 84% (95% CI; 81–88%). STEC isolated by Gastrointestinal Bacteria Reference Unit (GBRU) and stx2 and eae positive predicted HUSEC with a diagnostic accuracy of 99% (95% CI; 98–100%). Risk assessment combining these two tests predicts the most efficient use of resources, predicting that 18% (97/552) of cases would be eligible for follow-up at some stage, 16% (86/552) following local stx PCR results, 1% (7/552) following GBRU results of stx2 and eae status and 0.7% (4/552) following whole-genome sequencing. Follow-up could be stopped in 78% (76/97) of these cases, 97% (74/76) following second stage risk assessment. Conclusions This three-stage risk assessment approach prioritizes follow-up to HUSEC and minimizes unnecessary public health actions. We developed it into the algorithm for public health actions included in the updated PHE Guidance for management of STEC published in August 2018.

Resistance of Shiga toxin-producing Escherichia coli (STEC) O157:H7 and serogroups O103, O26 and O145 to synthetic gastric fluid (SGF, pH 1.5) were investigated during frankfurter storage. Pathogens were inoculated (5 ± 1 log(10 )cfu g(−1)) on frankfurters and frankfurters were stored at 4 °C for 75 days in vacuum packages. Population changes of the competitive flora and STEC, changes in the pH of the frankfurters and resistance of STEC to SGF were monitored on days 0, 15, 30, 45, 60 and 75 of frankfurter storage. Direct synthetic gastric fluid (DSGF) challenges were also conducted to assess pathogen resistance without being effected by frankfurters, by inoculating pathogen cultures directly into SGF. Results showed that acid resistance of O145 and O26 was stronger than that of O103 and O157 during frankfurter storage. Resistance of O103 to SGF was better than that of O157 during frankfurter storage but, was similar to that of O157 during DSGF challenges. Results indicate that acid resistance of some strains of STEC pathogens might differentiate during storage of frankfurters. Different resistance capabilities to SGF were observed in the STEC strains when inoculated and stored on frankfurters than directly inoculated in the SGF.

Computational method can be used for investigation of the protein sequences for developing a vaccine against infections. In this present study, a protein derived from non-O157 Verotoxin-producing E. coli (VTEC) was identified as a potential vaccine candidate that can be used to evaluate their immunogenicity and protective capability against VTEC infections. Identification of potential B-cell epitopes for promising vaccine was carried out by evaluating the protein derived from non-O157 VTEC with the methods of beta turns, hydropathicity, surface accessibility and antigenicity. The methods were implemented in MATLAB. Our test results demonstrated that the VTEC-derived protein has plausible characteristics which provide significant insights for further investigations and will assist in finding potential drug targets/vaccine candidates.

Forty-eight Escherichia coli strains were chosen due to variable detection of stx or serogroup by PCR. Although all strains were initially determined to be Shiga toxin-producing Escherichia coli (STEC), their genomes revealed 11 isolates carrying stx1a, stx1b, stx2a, and/or stx2b. Assembled genome sizes varied between 4,667,418 and 5,556,121 bp, with N50 values between 79,648 and 294,166 bp and G+C contents between 50., Forty-eight Escherichia coli strains were chosen due to variable detection of stx or serogroup by PCR. Although all strains were initially determined to be Shiga toxin-producing Escherichia coli (STEC), their genomes revealed 11 isolates carrying stx1a, stx1b, stx2a, and/or stx2b. Assembled genome sizes varied between 4,667,418 and 5,556,121 bp, with N50 values between 79,648 and 294,166 bp and G+C contents between 50.3% and 51.4%.

Shiga toxin-producing Escherichia coli, especially E. coli O157 is an important foodborne pathogen capable ofcoexisting in multispecies biofilms found in almost all the natural environments. Biofilm cells are usually more resistant than planktonic cells against environmental stresses. Thus, E. coli O157 in biofilms is a serious food safety concern. This study describes the characterization of a bacteriophage FP43 isolated from bovine intestine and the ability of FP43 to inhibit and remove multi species biofilms of E. coli O157 strain 196 and non-O157 strain 104. Phage FP43 has a short latent period of 15 min and a large burst size of 98 PFU/cell, with great stability at temperatures ranging from 4 to 60°C and pH from 4 to 9. To evaluate the effects of FP43 on E. coli,in microplate, biofilm formation was determined by crystal violet stainingas well as viable counts of biofilm and planktonic cells by conventional plating method. Phage FP43 decreased biofilm adhesionof E. coli cells with equal proportions of E. coli O157 and non-O157 by 82.4%. Viable counts were also reduced by 2.76 and 2.85 log in E. coli O157 and total biofilm cells after 6-h infection, respectively, compared with control. In planktonic cells, E. coli O157 and total counts decreased by 3.44 and 3.62 log after a 4-h phage treatment, respectively. Moreover, after a 6-h exposure to phage FP43, more than 60% of established biofilms were removed, and E. coli O157 and total viable counts in biofilm were decreased by 2.07 and 1.93 log, respectively. These findings suggest that phage FP43 seems to be a potential agent against E. coli O157 in multi species biofilms.

Shiga toxin-producing Escherichia coli (STEC) is one of the main cause of foodborne disease worldwide, but isolation rates or characteristics of this bacteria from ground beef in Chile are unknown. The present study aimed to isolate and characterize non-O157 STEC from ground beef sold at retail in the city of Santiago, Chile. We analyzed 430 ground beef samples for the presence of STEC, and isolated the microorganism in 10% of samples (43/430). We obtained 56 isolates from the 43 positive samples; 55 of these (98.2%) fermented sorbitol. Most isolates (98.2%; 55/56) showed β-glucoronidase activity, and only six (10.7%; 6/56) were resistant to tellurite. Among the virulence factors studied (stx1, stx2, eae, and hlyA), stx2 was the only virulence factor in 41% of the isolates (23/56), whereas 10.7% (6/56) of isolates carried a combination of three virulence factors (stx1 + stx2 + hlyA). None of the isolates carried the gene eae. Finally, isolates were neither serogroups O157 nor “big six”. In conclusion, ground beef sold in Santiago, Chile is contaminated with STEC; however, further studies are required for understanding their virulence potential.

Shiga toxin-producing Escherichia coli (STEC) are globally important food-borne pathogens. The isolation of non-O157 STEC is a significant public health challenge due to the dramatic diversity of their phenotypes and genotypes. In the present study, 476 non-O157 STEC strains representing 95 different O-serogroups were used to evaluate tellurite resistance and the performance of 12 different chromogenic agars. Of 476 strains, only 108 (22.7%) strains showed the minimal inhibitory concentration (MIC) values for potassium tellurite being higher than 4μg/ml, and 96 (20.2%) strains harbored intact ter genes cluster. The presence of ter genes was significantly correlated with tellurite resistance. Six commercial chromogenic agars (TBX, MAC, SMAC, Rainbow® Agar O157, CHROMagar™ ECC, and Fluorocult O157) supported the growth of all strains. However, CT-SMAC, CHROMagar™ O157, and CHROMagar™ STEC agars exhibited 12.2%, 31.1%, and 38.0% of growth inhibition, respectively. Furthermore, 4.6%, 33.2%, and 45.0% of strains were inhibited on RBA-USDA, RBA-NT, and BCM O157 agar media. Variations in tellurite resistance and colony appearance might result in discrepant performance of non-O157 STEC recovery from different chromogenic agars. Using inclusive agars or less selective agar in combination with highly selective agar should be suggested to recover most non-O157 STEC strains, which would increase the probability of recovering STECs from complex background microflora.

Shiga toxin-producing Escherichia coli (STEC) cause illnesses ranging from mild diarrhea to ischemic colitis and hemolytic uremic syndrome (HUS), serogroup O157 is the most common cause. We describe the epidemiology and transmission routes for U.S. STEC outbreaks during 2010–2017. Health departments reported 466 STEC outbreaks affecting 4769 persons, 459 outbreaks had a serogroup identified (330 O157, 124 non-O157, 5 both). Among these, 361 (77%) had a known transmission route: 200 foodborne (44% of O157 outbreaks, 41% of non-O157 outbreaks), 87 person-to-person (16%, 24%), 49 animal contact (11%, 9%), 20 water (4%, 5%), and 5 environmental contamination (2%, 0%). The most common food category implicated was vegetable row crops. The distribution of O157 and non-O157 outbreaks varied by age, sex, and severity. A significantly higher percentage of STEC O157 than non-O157 outbreaks were transmitted by beef (p = 0.02). STEC O157 outbreaks also had significantly higher rates of hospitalization and HUS (p &lt, 0.001).

Dairy compost with 20, 30, or 40% moisture content (MC) was inoculated with a mixture of six non-O157 Shiga toxin-producing Escherichia coli (STEC) serovars at a final concentration of 5.1 log CFU/g and then stored at 22 and 4°C for 125 days. Six storage conditions-4°C and 20% MC, 4°C and 30% MC, 4°C and 40% MC, 22°C and 20% MC, 22°C and 30% MC, and 22°C and 40% MC-were investigated for the persistence of non-O157 STEC in the dairy compost. During the entire storage, fluctuations in indigenous mesophilic bacterial levels were observed within the first 28 days of storage. After inoculation, the non-O157 STEC population increased 0.69 and 0.79 log CFU/g in the dairy compost with 30 and 40% MC at 22°C within the first day, respectively; for all other storage conditions, the pathogen population decreased rapidly. After the 125-day storage, the reductions of non-O157 STEC for 4°C and 20% MC, 4°C and 30% MC, 4°C and 40% MC, 22°C and 20% MC, 22°C and 30% MC, and 22°C and 40% MC storage conditions were >4.52, >4.55, 3.89, >4.61, 3.60, and 3.17 log CFU/g, respectively. All the survival curves showed an extensive tail, indicating non-O157 STEC can survive at least for 125 days in the dairy compost. The survival data were analyzed with log-linear with tailing and Weibull models. Compared with the log-linear with tailing model, the Weibull model was found to be a better choice for predicting the survival of non-O157 STEC in dairy compost owing to a high overall R2 value (0.8738 to 0.9909). The decay rate of non-O157 STEC was higher in dairy compost stored at 4°C compared with at 22°C, and the same trend was found for the compost with 40% MC versus 20% MC. In addition, two non-O157 STEC serotypes (STEC O145 and O45) were detected on the last day of the longitudinal study and may deserve special attention in the Big 6 STEC group. Our results have provided scientific data for risk assessment of the microbiological safety of dairy compost to control non-O157 STEC during subsequent storage of dairy compost.

The study examined the relative fate of the top six non-O157 shiga-toxin producing Escherichia coli (STEC) and E. coli O157:H7 during the manufacture of dry fermented sausages (DFS). Three separate batches of sausages containing a five-strain cocktail for each serogroup and uninoculated control were manufactured and subjected to identical fermentation, maturation and dry curing conditions. Changes in physicochemical properties and inoculated STEC numbers were enumerated during the DFS production stages and log reduction and log reduction rates were calculated. Inoculation of very high concentrations (8logCFUg-1) of STEC in the sausage batter did not significantly (P>0.05) affect the changes in the pH, aw, moisture, protein, fat content compared to the uninoculated DFS. There was a significant (P 0.05) from each other. These results indicate that the lethality of DFS production processes observed against E. coli O157:H7 would result in a similar inactivation of the top six non-O157 STEC.

Immunomagnetic separation used with culture based methods has been a useful technique in the detection of pathogens. However, previous studies have not answered many of the necessary questions for real world applications. The objective of this study was to assess the efficacy of different immunomagnetic separation (IMS) bead types in recovery of the correct serogroup from a mixture of big six non-O157 Shiga toxin-producing Escherichia coli strains. To determine the impact of different matrices on recovery, samples of sterile phosphate buffered saline (PBS), sterile and non-sterile cattle faeces, ground beef and lettuce were inoculated with 10 CFU per ml mixture of isolates representing the six serogroups. After a 6 h incubation at 37°C, samples were mixed with IMS beads from three different commercial sources and plated on eosin methylene blue agar (EMB). Three suspect E. coli colonies were selected from each EMB plate and multiplex polymerase chain reaction was used to determine the serogroup. The rate of correct identification varied with the serogroup, IMS bead manufacturer and matrix. Overall, recovery of the correct serogroup became less likely with increase in matrix complexity, with enrichments containing lettuce having the greatest number of bead types with significantly lower likelihood of correct recovery compared to recovery in PBS. Significance and Impact of the Study The need to accurately and efficiently detect Shiga toxin-producing Escherichia coli (STEC) O26, O45, O103, O111, O121 and O145, which have caused outbreaks on numerous occasions, is a major public health and food safety concern in the United States. Detecting these STEC serogroups can be challenging because methods to detect non-O157 serogroups have not been refined as compared to those for O157. Immunomagnetic separation (IMS) has the potential to isolate STEC from a mixture in complex matrices. Our results highlight the need for optimization of IMS-based detection of STEC to effectively recover the targeted serogroup from a variety of sample matrices.

Six major Shiga toxin producing Escherichia coli (STEC) serogroups: O26, O103, O145, O111, O121, and O45 have been declared as adulterants in federally inspected raw beef in the USA effective June 4th, 2012 in addition to the routinely tested STEC O157: H7. This study tests a real-time multiplex PCR assay and pooling of the samples to optimize the detection and quantification (prevalence and contamination) of six major non-O157 STEC, regardless of possessing Shiga toxins. To demonstrate the practicality, one large-scale slaughter plant (Plant LS) and one small-scale slaughter plant (Plant SS) located in the Mid-Western USA were sampled, in 2011, before the establishment of 2013 USDA laboratory protocols. Carcasses were sampled at consecutive intervention stations and beef trimmings were collected at the end of the fabrication process. Plant SS had marginally more contaminated samples than Plant LS (p-value 0.08). The post-hide removal wash, steam pasteurization, and lactic acid (≤5%) spray used in Plant LS seemed to reduce the six serogroups effectively, compared to the hot-water wash and 7-day chilling at Plant SS. Compared to the culture isolation methods, quantification of the non-O157 STEC using real-time PCR may be an efficient way to monitor the efficacy of slaughter line interventions.

Summary Non-O157 Shiga toxin-producing Escherichia coli (STEC) are a frequent cause of STEC-related infections such as diarrhoea. Fermentation by presumptive probiotic Lactobacillus plantarum strain B411 isolated from cereal fermentation was investigated to prevent the growth of acid-adapted (AA) and non-acid-adapted (NAA) non-O157 STEC in traditionally fermented maize gruel, a widely used complementary food in Africa. L. plantarum strain B411 possessed probiotic characteristics and antimicrobial activity against selected pathogenic bacteria. Growth of AA and NAA non-O157 STEC strains was substantially inhibited by 3.6 and 4.8 log reductions, respectively, in the maize gruel fermented with the L. plantarum B411, while their growth was only inhibited by 1.0 and 1.2 log reductions, respectively, by traditional fermentation alone. Inclusion of fermentative strains of L. plantarum exhibiting probiotic activity is a feasible method to ensure safety of traditionally fermented African cereal porridges through inhibition of non-O157 STEC.

We investigated for virulence genes ( stx1 , stx2 and hlyA ), serotypes and extended-spectrum β-lactamases (ESBLs) producing capacity in O157 and non-O157 Escherichia coli isolated from producer-distributor bulk milk (PDBM). Fifteen different E. coli O-serogroups were observed from the isolates (n = 121). The prevalence of stx1 and stx2 genes among the E. coli isolates was 8.3% and 11.6%, respectively, while 5.8% harboured both stx1 and stx2 . Four E. coli isolates (3.3%) had ESBLs producing capacity, resisted multiple cephalosporins and aztreonam, and carried stx genes. Cluster analysis using GTG 5 finger printing revealed a diversity of E. coli seropathotypes in PDBM that are known to be associated with human diarrhoeal diseases. These results highlight a potential risk posed on human health by the consumption of PDBM contaminated with pathogenic E. coli . A further quantitative risk assessment of the impact of pathogenic E. coli contamination in PDBM on human health is therefore recommended.

Strawberries are vulnerable to harboring microbial pathogens because they are generally not washed due to their perishable nature. The focus of this study was to quantify the reduction in infection risks associated with non-O157 Shiga toxin producing Escherichia coli serotypes contaminated strawberries if the strawberries are exposed to low doses ∼1 kGy (kiloGray) of electron beam (eBeam) irradiation. A cocktail of six serotypes of non O157 E. coli namely, O26:H11, O45:H2, O103:H2, O111:NM, O121:H19, and O145 was employed. Strawberry puree rather than whole strawberries were used in this study to ensure dose uniformity that is critical for accurate interpretation of microbial reduction. The results show that when these serotypes are exposed to ≤1 kGy eBeam dose, there is approximately 4-log reduction in their numbers when they are present within a strawberry matrix (puree). Quantitative microbial risk assessments suggest that if a typical strawberry serving (150 g) was heavily contaminated (∼105 CFU/serving size), 2 out of 10 susceptible individuals (20%) would get sick (without eBeam treatment). However, if these contaminated strawberries had been treated with 1 kGy of eBeam dose, the infection risks would have be significantly reduced to approximately 4 out of every 100,000 individuals (0.004%). Similarly, even at low levels of contamination (∼102 CFU/serving), the infection risks would be reduced from 6 out of 10,000 susceptible individuals to approximately 4 out of 100 million susceptible individuals.

Verocytotoxin-producing E. coli (VTEC) of serotype O157:H7 have been shown to be important agents of foodborne disease in humans worldwide. While the majority of research effort has been targeted on this serotype it is becoming more evident that other serotypes of VTEC can also be associated with human disease. An increasing number of these non-O157:H7 VTEC have been isolated from humans suffering from HUS and diarrhea. Recently a number of foodborne outbreaks in the USA, Australia, and other countries have been attributed to non-O157:H7 VTEC serotypes. Surveys of animal populations in a variety of countries have shown that the cattle reservoir contains more than 100 serotypes of VTEC, many of which are similar to those isolated from humans. The diversity and complexity of the VTEC family requires that laboratories and public health surveillance systems have the ability to detect and monitor all serotypes of VTEC.

Shiga toxin-producing Escherichia coli (STEC) are important pathogens transmitted by food that may cause severe illness in human beings. Thus, systems for STEC detection in food should have increasingly higher sensitivity and specificity. Here we compared six commercial systems for non-O157 STEC detection in meat and vegetables and determined their sensitivity, specificity and repeatability. A total of 46 samples (meat n=23; chard n=23) were experimentally contaminated with strains O26:H11, O45:H-, O103:H2, O111:NM, O121:H19 and O145:NM isolated in Argentina. Strain detection was confirmed by isolation according to ISO 13136:2012. Detection of the stx and eae genes in meat samples was highly satisfactory with all commercial kits, but only five had 100% sensitivity and specificity in chard. Of four kits evaluated for serogroup detection, three had 100% sensitivity and specificity, and one had 93.7% sensitivity and 100% specificity. All kits were adequate to analyze meat but not vegetable samples, and were not therefore validated for the latter matrix. The challenge for microbiology laboratories is to identify the advantages and disadvantages of the available kits for STEC detection in food based on a clear knowledge of the particular needs of each laboratory. Fil: Costa, Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina Fil: Sucari, Adriana. Stamboulian Servicios de Salud; Argentina Fil: Epszteyn, Sergio. No especifíca; Fil: Oteiza, Juan Martín. Instituto Nacional de Tecnologia Industrial. Centro de Investigacion y Asistencia Tecnica A la Industria Villa Regina.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gentiluomo, Jimena. No especifíca; Fil: Melamed, Celia. No especifíca; Fil: Figueroa, Yamila. Stamboulian Servicios de Salud; Argentina Fil: Mingorance, Santiago. No especifíca; Fil: Grisaro, Agustina. Stamboulian Servicios de Salud; Argentina Fil: Spioussas, Silvia. No especifíca; Fil: Buffoni Almeida, Mariana. Stamboulian Servicios de Salud; Argentina Fil: Caruso, Mailen. No especifíca; Fil: Pontoni, Andrés. No especifíca; Fil: Signorini, Marcelo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina Fil: Leotta, Gerardo Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina

Background: The Polyskope 1.0 Multiplex Assay is a novel test to simultaneously detect Escherichia coli O157, non-O157 Shiga Toxin-Producing E. coli (STEC), Listeria monocytogenes, and Salmonella species in a single enrichment using real-time PCR. Objective: A Performance Tested MethodSM study was conducted to validate Polyskope 1.0 for inclusivity and exclusivity as well as a matrix comparison study. Method: This assay was evaluated in an unpaired independent validation study compared with reference methods according to AOAC INTERNATIONAL validation guidelines. Polyskope 1.0 evaluated raw ground beef (25 g), deli turkey (25 g), baby spinach (25 g), and stainless-steel environmental surface sponges (4 × 4 in. test area) after inoculation with a suspension of the three target microorganisms. All matrices were compared with appropriate reference methods from the U.S. Food and Drug Administration Bacteriological Analytical Manual, U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook, or International Organization for Standardization standards. Results: Polyskope 1.0 demonstrated no statistically significant differences between candidate and reference method results or between presumptive and confirmed results for three food matrices and one environmental surface. Results from inclusivity and exclusivity evaluations indicated the test method can accurately detect the target analytes and excluded all nontarget organisms. No differences were observed with the stability or lot-to-lot evaluations. Polyskope 1.0 demonstrated robustness by remaining unaffected by small variations in method parameters, which had no statistically significant effect on the results for all eight variations. Conclusions and Highlights: Polyskope 1.0 was shown to be a specific, highly accurate, and robust method for the detection of Listeria monocytogenes, Salmonella species, non-O157 STECs, and E. coli O157 across four matrices.

Shiga toxin-producing Escherichia coli (STEC) serogroups O26, O45, O103, O111, O121, and O145, referred to as the top six non-O157 serogroups, are responsible for more than 70% of human non-O157 STEC infections in North America. Cattle harbor non-O157 strains in the hindgut and shed them in the feces. The objective of this study was to use the U.S. Food and Drug Administration (FDA) E. coli identification (ECID) DNA microarray to identify the serotype, assess the virulence potential of each, and determine the phylogenetic relationships among five of the six non-O157 E. coli serogroups isolated from feedlot cattle feces. Forty-four strains of STEC, enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), or putative nonpathotype E. coli (NPEC) of cattle origin and five human clinical strains of EHEC were assayed with the FDA-ECID DNA microarray. The cattle strains harbored diverse flagellar genes. The bovine and human strains belonging to serogroups O26, O45, and O103 carried stx

peer-reviewed Phenotypic, chemotaxonomic and genotypic data from 12 strains of Escherichia coli were collected, including carbon source utilisation profiles, ribotypes, sequencing data of the 16S–23S rRNA internal transcribed region (ITS) and Fourier transform-infrared (FT-IR) spectroscopic profiles. The objectives were to compare several identification systems for E. coli and to develop and test a polyphasic taxonomic approach using the four methodologies combined for the sub-typing of O157 and non-O157 E. coli. The nucleotide sequences of the 16S–23S rRNA ITS regions were amplified by polymerase chain reaction (PCR), sequenced and compared with reference data available at the GenBank database using the Basic Local Alignment Search Tool (BLAST) . Additional information comprising the utilisation of carbon sources, riboprint profiles and FT-IR spectra was also collected. The capacity of the methods for the identification and typing of E. coli to species and subspecies levels was evaluated. Data were transformed and integrated to present polyphasic hierarchical clusters and relationships. The study reports the use of an integrated scheme comprising phenotypic, chemotaxonomic and genotypic information (carbon source profile, sequencing of the 16S–23S rRNA ITS, ribotyping and FT-IR spectroscopy) for a more precise characterisation and identification of E. coli. The results showed that identification of E. coli strains by each individual method was limited mainly by the extension and quality of reference databases. On the contrary, the polyphasic approach, whereby heterogeneous taxonomic data were combined and weighted, improved the identification results, gave more consistency to the final clustering and provided additional information on the taxonomic structure and phenotypic behaviour of strains, as shown by the close clustering of strains with similar stress resistance patterns. The authors acknowledge the financial contribution of the Spanish INIA, the Research Council of Norway (project 178230/I10), Foundation for Levy on Foods, the Norwegian Research Fees Fund for Agricultural Goods, the Norwegian Independent Meat and Poultry Association, Nortura SA and NHO Matog Landbruk.

Escherichia coli O157:H7 has frequently been associated with foodborne infections and is considered an adulterant in raw non-intact beef in the U.S. Shiga toxin-producing E. coli (STEC) belonging to serogroups O26, O45, O103, O111, O121, and O145 (known as the “big six” non-O157) were estimated to cause > 70% of foodborne infections attributed to non-O157 serogroups in the U.S., as a result, these six serogroups have also been targeted by regulation in the U.S. The purpose of this study was to develop a rapid and high-throughput molecular method to group STEC isolates into seven clinically important serogroups (i.e., O157 and the “big six” non-O157 serogroups) targeted by regulation in the U.S. by interrogating single nucleotide polymorphisms (SNPs) in gnd. A collection of 195 STEC isolates, including isolates belonging to O157:H7 (n = 18), O26 (n = 21), O45 (n = 19), O103 (n = 24), O111 (n = 24), O121 (n = 23), O145 (n = 21), and ten other STEC serogroups (n = 45), was assembled and characterized by full gnd sequencing to identify informative SNPs for molecular serogrouping. A multiplex SNP typing assay was developed to interrogate twelve informative gnd SNPs by single base pair extension chemistry and used to characterize the STEC isolate collection assembled here. SNP types were assigned to each isolate by the assay and polymorphisms were confirmed with gnd sequence data. O-serogroup-specific SNP types were identified for each of the seven clinically important STEC serogroups, which allowed the differentiation of these seven STEC serogroups from other non-O157 STEC serogroups. Although serogroups of the “big six” non-O157 STEC and O157:H7 contained multiple SNP types per O-serogroup, there were no overlapping SNP types between serogroups. Our results demonstrate that molecular serogrouping of STEC isolates by interrogation of informative SNPs in gnd represents an alternative to traditional serogrouping by agglutination for rapid and high-throughput identification of clinically important STEC serogroups targeted by regulation for surveillance and epidemiological investigations.

In this study, specific and rapid enrichment and growth conditions for the most important, classic non-O157 enterohemorrhagic Escherichia coli (EHEC) serogroups were assessed. Three enrichment broth types, namely, EC medium with MUG broth, BRILA broth, and mTSB broth with novobiocin, were compared to identify the optimum enrichment broth for EHEC isolation. Four kinds of selective media, namely, ENDO agar, Chromocult agar, TBX agar, and CHROMagar TM STEC medium, were compared to identify the optimum one for non-O157 EHEC isolation. The results suggested that incubation in EC medium with MUG broth at 42℃ for 20 h is optimum for the enrichment of non-O157 EHEC. TBX agar was identified to have the highest specificity among the tested media. Consequently, a combination of complementary selective media according to serotype must be considered for comprehensive isolation of specific EHEC.

This study was conducted to investigate the presence of shiga toxin producing Escherichia coli (STEC) and the possibility of carrying rfbE gene and H7 flagellar on meat, liver, and stool samples collected from Jakarta Province of Indonesia. A total of 51 samples collected from meat, liver, and stool of slaughtered cattle from qurban festival were tested using conventional culture and multiplex PCR methods. STEC non O157 were detected in meat (5.3%) and stool (8.3%) with one isolate from stool carried H7 flagellar. However, all isolates were lacking of rfbE gene. In antimicrobial susceptibility tests, the STEC isolates showed antibiotic resistance to erythromycin and oxacillin. Overall, the result shows that meat and liver of this origin activity represents a potential risk to human health.

Detection of Shiga toxin-producing Escherichia coli (STEC) has evolved significantly since the introduction of sorbitol-MacConkey agar. This study compares four chromogenic media (CHROMagar™ STEC, Rainbow® O157 agar, CHROMagar™ O157, and Colorex® O157) in their identification of non-O157 STEC. When 161 non-O157 STEC were directly inoculated onto each medium, detection rates on CHROMagar™ STEC, Rainbow® O157 agar, CHROMagar™ O157 and Colorex® O157 were 90%, 70%, 3.7% and 6.8%, respectively. Tellurite minimal inhibitory concentrations (MICs) correlated with growth on CHROMagar™ STEC as 20 of 22 isolates with poor or no growth had MICs ≤1μg/mL. Stool spiking experiments revealed that CHROMagar™ STEC had the highest recovery of the six most common non-O157 STEC, ranging from 30% (in mucoid stool) to 98% (in watery stool). When using clinical stool samples, CHROMagar™ STEC had a sensitivity, specificity, positive predictive value, and negative predictive value of 84.6%, 87%, 13.9%, and 99.6%, respectively.

The goal of this study was to evaluate the efficacy of a novel antimicrobial solution made with chitosan, lauric arginate ester, and organic acids on Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes, and non-O157 shiga toxin-producing E. coli cocktails and to test its potential to be used as a marinade for raw beef. Fresh beef top round steaks were surface-inoculated with the pathogen cocktails at approximately 2.5 or 4.5 Log CFU/cm2, marinated with the antimicrobial solution (AMS), and then stored at 4 °C for 6, 24, and 48 h. Three commercially available marinades were used for comparison. Results revealed that AMS had the most antimicrobial effect regardless of the type or inoculation level of pathogens (P

Shiga toxin-producing Escherichia coli (STEC) is one of the most significant causes of food-borne infections capable of causing serious health complications in humans. Even though ruminants are known to be the major reservoirs of STEC, other non-ruminant food producing animals may also harbour pathogenic E. coli strains. In this study, we investigated the prevalence of E. coli serogroups O26, O111, O121, O145, and O157 and their associated virulence genes (stx1, stx2, eae, and ehxA) in swine faecal samples obtained from the two major commercial farms located in the Nkonkobe Municipality, Eastern Cape, South Africa. The proportions of serogroups detected were O26; 35 (7%), O145; 14 (2.8%), and O157:H7; 43 (8.6%) of the total animals sampled. Out of the 500 animals sampled, 22 isolates of E. coli (1.4%) tested positive for the stx2 gene, and 7 of these isolates belonged to E. coli O26 serogroup, while the remaining 15 most likely belonged to serogroups untargeted in this study. Other virulence genes (stx1, eae, and ehxA) that we screened for were not detected. These findings reveal that pigs within the Eastern Cape Province of South Africa can harbour Shiga toxin-producing E. coli.

Latex agglutination tests (LAT) were developed and evaluated for the rapid detection of LPS antibodies against E. coli serogroup O157, O26, O104, O111 and O145. The latex tests have been proved to be sensitive, fast, easy-to-perform and cost-efficient tools for the screening serodiagnosis of VTEC infections causing haemolytic-uraemic syndrome.

We compared 47 enterohemorrhagic Escherichia coli (EHEC) O157 isolates with 184 EHEC non-O157 isolates from Korean patients with diarrhea. In the O157 group, the strains harboring both Shiga toxin genes (stx1 and stx2) were detected with highest frequency, whereas the strains harboring only stx1 gene were most frequently detected in the non-O157 group. Eight virulence genes (eaeA, hlyA, ehx, iha, efa1, tir, toxB, and espA) were found to show a higher frequency of occurrence in the O157 group than in the non-O157 group. In addition, the symptom of bloody diarrhea was exhibited at a higher rate in the O157 group (51.1%) than in the non-O157 group (16.8%). Our findings demonstrate that EHEC O157 strains are more frequently implicated in cases of bloody diarrhea in the Korean population than EHEC non-O157 strains.

According to the United States Food and Drug Administration (FDA) agency, bacteriophage solutions targeting the serotype O157:H7 are Generally Recognized as Safe (GRAS) to control STEC during beef processing. However, outbreaks involving the "Big Six" STEC increased the industry concern about those serotypes. The objective of this study was to test the efficacy of MS bacteriophages to reduce the "Big Six" non-O157 STEC in beef. The lysing efficacy of phages isolated for each specific serotype varied from 96.2% to 99.9% in vitro. When applied to contaminated trim, reductions ranging from 0.7 to 1.3 Log of all STEC were observed in ground beef. Bacteriophages may provide an additional barrier against the "Big Six" STEC in ground beef. Results of this research provide support documentation to the FDA to extend GRAS status for bacteriophages as processing aids against all adulterant STEC.

This study was carried out to assess the survival of Shiga toxin-producing E. coli (STEC) and atypical enteropathogenic Escherichia coli (aEPEC) during the traditional manufacturing and ripening of Spanish hard cheese from raw cow&rsquo, s milk. Milk samples were spiked with up to 3.1&ndash, 3.5 log cfu/mL of one strain of STEC (O140:H32 serotype) and one of aEPEC (serotype O25:H2). The first steps of cheesemaking allow for a STEC and aEPEC increase of more than 1 log cfu/mL (up to 4.74 log cfu/g and 4.55 log cfu/g, respectively). After cheese pressing, a steady reduction of both populations was observed, with the STEC strain being more sensitive. The studied pathogenic E. coli populations decreased by 1.32 log cfu/g in STEC and 0.59 log cfu/g in aEPEC in cheese ripened during a minimum period of 60 d. Therefore, a moderate contamination by these diarrhoeagenic E. coli pathotypes, in particular, with aEPEC, on cheese manufactured from raw milk may not be totally controlled through the cheesemaking process and during a maturation of 90 d. These findings remark the importance of improvement in bacteriological quality of raw milk and cross-contamination prevention with diarrhoeagenic E. coli in the dairy industry.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups such as O26, O45, O103, O111, O121 and O145 often cause illness to people in the United States and the conventional identification of these “Big-Six” are complex. The label-free hyperspectral microscope imaging (HMI) method, which provides spectral “fingerprints” information of bacterial cells, was employed to classify serogroups at the cellular level. In spectral analysis, principal component analysis (PCA) method and stacked auto-encoder (SAE) method were conducted to extract principal spectral features for classification task. Based on these features, multiple classifiers including linear discriminant analysis (LDA), support vector machine (SVM) and soft-max regression (SR) methods were evaluated. Different sizes of datasets were also tested in search for the suitable classification models. Among the results, SAE-based classification models performed better than PCA-based models, achieving classification accuracy of SAE-LDA (93.5%), SAE-SVM (94.9%) and SAE-SR (94.6%), respectively. In contrast, classification results of PCA-based methods such as PCA-LDA, PCA-SVM and PCA-SR were only 75.5%, 85.7% and 77.1%, respectively. The results also suggested the increasing number of training samples have positive effects on classification models. Taking advantage of increasing dataset, the SAE-SR classification model finally performed better than others with average accuracy of 94.9% in classifying STEC serogroups. Specifically, O103 serogroup was classified with the highest accuracy of 97.4%, followed by O111 (96.5%), O26 (95.3%), O121 (95%), O145 (92.9%) and O45 (92.4%), respectively. Thus, the HMI technology coupled with SAE-SR classification model has the potential for “Big-Six” identification.